Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/20—Carbocyclic rings
  • C07H15/22—Cyclohexane rings, substituted by nitrogen atoms
  • C07H15/222—Cyclohexane rings substituted by at least two nitrogen atoms
  • C07H15/226—Cyclohexane rings substituted by at least two nitrogen atoms with at least two saccharide radicals directly attached to the cyclohexane rings
  • C07H15/234—Cyclohexane rings substituted by at least two nitrogen atoms with at least two saccharide radicals directly attached to the cyclohexane rings attached to non-adjacent ring carbon atoms of the cyclohexane rings, e.g. kanamycins, tobramycin, nebramycin, gentamicin A2

Definitions

• the invention relates to an unobvious improved process for the isolation and purification of aminoglycoside antibiotics of the formula ##STR1## wherein X denotes a radical of the formula ##STR2## Y denotes a radical of the formula ##STR3## R denotes a hydrogen atom or an ethyl group, R 1 denotes a C 1 to C 6 alkyl group, preferably a methyl or ethyl group, and
• one of the radicals Z or W denotes a hydrogen atom
• the other radical Z or W denotes a hydrogen atom or a hydroxyl group.
• the process according to the invention is preferably used for the purification or preparation of sisomicin, 5-episisomicin, netilmicin, gentamicin and 3"-N-demethyl-3"-N-ethylsisomicin.
• the compounds of the formula (I) are known aminoglycoside antibiotics. Some of them are obtained biosynthetically by culturing, in an aqueous nutrient medium under specific conditions, a strain which produces the antibiotic in question, or by chemical modification of an antibiotic obtained by the abovementioned biosynthetic process.
• the present invention relates to a multi-stage working-up process which utilises a new, advantageous concept of isolating and purifying aminoglycosides; it combines selective lipophilisation of the particular compound of the formula (I) in the crude product obtained by fermentation with controlled liquid/liquid extraction of this lipophilised product.
• the lipophilisation is achieved by providing the amino groups contained in the compounds of the formula (I) with suitable protective groups. After the desired aminoglycoside derivative has been separated off in a pure form by liquid/liquid extraction, the protective groups are split off again and the desired aminoglycoside of the formula (I) is obtained in a pure form.
• the fermentation broth is pre-purified and the amino groups of the desired compound and of the undesired concomitant substances are reacted with protective group reagents selected for the individual case.
• the pre-purified fermentation broth of a biosynthetically produced aminoglycoside is likewise used as the starting material, the subequent step being a selective reaction with protective group reagents selected for the individual case.
• the desired chemical reaction for example the introduction of an ethyl group into the 1-position or the replacement of the methyl group in the 3"-position by an ethyl group, is carried out using the selectively blocked intermediate products thus obtained, this being followed, if appropriate, by a further reaction with protective group reagents.
• a pre-purified compound of formula (I), as defined previously, is acylated or arylsulphenylated to give a compound of the formula ##STR4## wherein X' represents a radical of the formulae ##STR5## Y' represents a radical of the formulae ##STR6## the radicals R 2 are identical or different and represent a hydrogen atom or an acyl or arylsulphenyl protective group, with the proviso that at most two of the radicals R 2 represent hydrogen atoms, and R, R 1 , Z and W have the abovementioned meaning, and optionally, where at least one of the radicals
• R 2 denotes a hydrogen atom, a chemical derivative is formed on one or more of the unprotected amino groups, (b) the compound of formula (II) from step (a) is subjected to liquid/liquid extraction in a two-phase aqueous/organic solvent system and is isolated from the extracts,
• radicals R 2 denotes a hydrogen atom
• a chemical derivative is formed on one or more of the unprotected amino groups, and (c) the protective group(s) are split off.
• pure used herein means that the aminoglycoside antibiotic is of sufficient purity for direct pharmaceutic use and, includes the isolation, if desired, of component of aminoglycoside antibiotic mixtures, whereas "pre-purified” merely refers to the removal of the major contaminants from the prior preparative process.
• acyl or arylsulphenyl protective group suitable for the individual case and the liquid/liquid extraction system can easily be established by preliminary experiments, with the assistance of suitable analytical methods, preferably by Craig distribution or high pressure liquid chromatography (HPLC).
• sisomicin (antibiotic 66-40) is purified in the following manner, as described in DE-OS (German Published Specification) No. 1,932,309, Examples 2 to 5:
• the fermentation broth is acidified, the mycelium is filtered off and the filtrate is neutralised with aqueous ammonia and, after removal of the calcium ions, treated with a cation exchanger resin, whereby the desired aminoglycoside is bonded, in addition to a considerable proportion of other organic and inorganic compounds.
• the resin is washed with water and subsequent elution with aqueous ammonia gives sisomicin as a 50% strength crude product.
• This product is further purified by ion exchanger chromatography and converted into the sulphate by treatment with sulphuric acid, and this sulphate is further purified by column chromatography and recrystallisation and finally converted back into the free base.
• the yield is less than 10%, relative to the 50% strength crude product.
• stage (a) of the process according to the invention is known, for example from European Published Patent Application 57.
• Suitable aryl or arylsulphenyl protective groups are known, for example from Houben-Weyl, Methoden der organischen Chemie (Methods of Organic Chemistry), Volume XV, Georg Thieme Verlag, Stuttgart, 1974.
• Preferred examples of such protective groups are acyl groups of the general formula ##STR7## wherein R 3 and R 4 independently denote a hydrogen atom or an optionally substituted phenyl radical and
• n, n 1 , n 2 and n 3 independently of one another denote 0, 1, 2, 3, 4 or 5,
• R 5 denotes an optionally substituted phenyl or di- or tri-phenylmethyl radical.
• R 3 , R 4 or R 5 preferably denotes a phenyl radical which is optionally monosubstituted or disubstituted by nitro, C 1 -C 4 -alkoxy, halogen or phenyl.
• the unprotected pre-purified compound of formula (I) is reacted in stage (a) with, for example, a compound of the following formula ##STR8## wherein G 1 denotes a halogen atom or another acylation reaction-leaving group, preferably a group which activates an ester,
• G 2 denotes a halogen atom or another sulphenylation reaction-leaving group, preferably a group which activates an ester, and
• R 3 , R 4 , R 5 , n, n 1 , n 2 and n 3 have the abovementioned meaning.
• the introduction of the protective groups is generally carried out, starting from the crude products obtained by fermentation, in an inert solvent at a temperature from -30° C. and +50° C., preferbly between 0° C. and 25° C., and if appropriate in the presence of a base.
• the reaction product is then worked up in the customary manner.
• Radical G 2 is preferably a chlorine atom or a p-nitrophenoxy radical.
• sulphenylating reagents which may be mentioned are tritylsulphenyl chloride, o-nitrophenylsulphenyl chloride, 2,4-dinitrophenylsulphenyl chloride, 2,4,5-trichlorophenylsulphenyl chloride, pentachlorophenylsulphenyl chloride, o-nitrophenylsulphenic acid p-nitrophenyl ester, 2,4-dinitrophenylsulphenic acid p-nitrophenyl ester, 2,4,5-trichlorophenylsulphenic acid p-nitrophenyl ester and pentachlorophenylsulphenic acid p-nitrophenyl ester.
• acylating reagents which may be mentioned are acetic anhydride, acetyl chloride and diethyl pyrocarbonate, the use of dialkylpyrocarbonates as protective group reagents in general being particularly preferred for the preparation of N-alkyl-oxycarbonyl derivatives.
• ossible diluents for the reaction with sulphenic acid halides are either inert organic solvents, such as chloroform and toluene, or, preferably, water-miscible solvents, such as dioxane, dimethylformamide and dimethoxyethane, and mixtures thereof with water.
• the reactions with activated esters of the above-mentioned sulphenic acids are preferably carried out in inert organic solvents, such as chloroform, dimethylformamide or pyridine, or mixtures of such solvents with alcohols, preferably methanol or ethanol.
• inert organic solvents such as chloroform, dimethylformamide or pyridine, or mixtures of such solvents with alcohols, preferably methanol or ethanol.
• the acyl compounds according to the invention are prepared in any desired inert organic solvents, in water or in mixtures of organic solvents and water, mixtures of methanol, ethanol or acetone and water being preferred.
• the sulphenylation or acylation reactions can be carried out either under normal pressure or under increased pressure. In general, the reactions are carried out under normal pressure.
• stage (a) of the process according to the invention for various aminoglycosides, are described below by way of example.
• sisomicin for example, a procedure can be followed in which acetic anhydride is added dropwise, at 5° C., to an aqueous crude sisomicin solution containing about 10% of sisomicin base. The sisomicin present in the solution and also the remaining amino compounds are thereby acylated on the primary amino groups. A mixture which contains 1,3,2',6'-tetra-N-acetylsisomicin is obtained in this manner.
• a crude gentamicin product reacts under the same conditions to give a mixture which contains 1,3,2',6'-tetra-N-acetylgentamicin C 1a. If pyrocarbonic acid dimethyl ester or diethyl ester is used as the protective group reagent and crude sisomicin is used as the substrate, 1,3,2',6'-tetra-N-methoxycarbonylsisomicin or -ethoxycarbonylsisomicin is formed in aqueous alcohol at low reaction temperatures. Other compounds which are present in the crude product employed and carry NH 2 groups likewise react, at the amino groups, with the pyrocarbonic acid esters, the corresponding urethanes being formed.
• the amino groups that is to say the primary and secondary amino groups with protective groups; the protective group reagents mentioned previously again being preferably used.
• This reaction is carried out in an inert solvent or in solvent mixtures, if appropriate with the addition of water, at temperatures between 0° and 100° C., preferably between 25° and 70° C., and if appropriate in the presence of a base. Since the secondary amino groups are in general less reactive than the primary amino groups, it is expedient to use an excess of protective group reagent; the water content of the solvent mixture used is kept as low as possible and, if necessary, the reacton is carried out at elevated temperature.
• Possible solvents for the reactions in question are, in principle, any solvents which dissolve the starting materials and either do not themselves react with the protective group reagents in question, or react with the protective group reagents in question with more difficulty than with the amino groups to be reacted. Examples which may be mentioned are chloroform, methylene chloride, ethanol, methanol, acetone and dioxane. Possible auxiliary bases are the compounds already mentioned. If, for example, the 1,3,2',6'-tetra-N-acetylsisomicin already mentioned is reacted with isopropylchloroformate at about 60° C.
• the lipophilic or hydrophilic character of the compounds of the formula (II) can be steered in a controlled manner such that an optimum ease of separation of undesired by-products by liquid/liquid extraction is achieved.
• the extraction systems used according to the invention are aqueous-organic solvent systems.
• a procedure is followed in which, after the extraction, the desired amino-trisaccharide derivative is in the organic phase.
• a suitable aqueous phase is, above all, aqueous ammonium hydroxide, for example a saturated solution of ammonia in water or solutions with a lower percentage content of ammonium hydroxide.
• ammonium hydroxide it is also possible to add other basic auxiliaries, such as organic amines, for example methylamine, dimethylamine and trimethylamine, to the aqueous phase.
• inorganic compounds such as alkali metal hydroxides, can also be used as basic auxiliaries.
• Suitable organic phases are solvents which are water-immiscible or only slightly water-miscible and are inert towards the product mixtures employed in the extraction, for example hydrocarbons (such as toluene, xylene, ethylbenzene and hexane), chlorinated hydrocarbons, (such as methylene chloride, 1,2-dichloroethane and 1,2-dichloropropane), esters (such as ethyl acetate, ethyl propionate and tributyl phosphate), ethers (such as diethyl ether), or alcohols (such as n-butanol, propan-2-ol and n-pentanol).
• hydrocarbons such as toluene, xylene, ethylbenzene and hexane
• chlorinated hydrocarbons such as methylene chloride, 1,2-dichloroethane and 1,2-dichloropropane
• organic phases which are to be particularly preferred are mixtures of water-immiscible solvents and other organic solvents, for example methylene chloride/propan-2-ol, methylene chloride/n-hexane, methylene chloride/n-butanol, 1,2-dichloroethane/n-hexane, butanol/n-hexane, methylene chloride/propan-2-ol/n-hexane and ethyl acetate/n-hexane.
• water-immiscible solvents and other organic solvents for example methylene chloride/propan-2-ol, methylene chloride/n-hexane, methylene chloride/n-butanol, 1,2-dichloroethane/n-hexane, butanol/n-hexane, methylene chloride/propan-2-ol/n-hexane and ethyl acetate/n-hexane
• the ratio of organic phase to aqueous phase can be varied within wide limits. This ratio is preferably 0.5:1 to 30:1, and very particularly preferably 5:1 to 20:1.
• the content of the desired amino-trisaccharide compound in the extract can be varied within wide limits by varying the amount of the extraction agent and the number of the extraction stages.
• the extraction agent employed is in general so chosen that extracts which contain the desired amino-trisaccharide derivative in a concentration of 0.1 to 15% are obtained.
• the amount of solvent in the aqueous crude solutions employed for the extraction is so chosen that a content of amino-trisaccharide derivative of between 1 and 20% results, 2 to 10% strength solutions preferably being employed.
• the percentage data are percentages by weight.
• the number of extraction stages necessary for optimum extraction is established in a known manner by preliminary experiments, for example via a distribution curve.
• the temperature of the extraction can be varied within wide limits. In general, the extraction is carried out at a temperature between 10° and 60° C. It can be carried out under normal pressure, reduced pressure or increased pressure. Possible extraction units are the known extraction systems, for example mixer/separators, centrifugal extractors or column systems, such as spray columns, stirred columns, such as Scheibel columns, or pulsating columns, such as pulsating perforated tray columns. In particularly simple cases, extractions can also be carried out by mixing and allowing to settle in one reaction vessel, for example in a stirred kettle. On a laboratory scale, it is also possible to use separating funnels, in the known manner, for carrying out the extraction. If columns are used, the extraction is preferably carried out as a countercurrent extraction.
• the extraction phase which contains the desired amino-trisaccharide derivative that is to say in general the organic phase, is concentrated or completely evaporated, in which case, in order to protect the product, those evaporator units in which the residence time of the product is as short as possible, for example, falling film evaporators or thin layer evaporators, are preferably chosen. Evaporation of the solvent is preferably carried out under reduced pressure.
• aminoglycoside antibiotics be separated off from their impurities, but also it is possible to separate aminoglycoside mixtures, such as are formed by culturing the corresponding micro-organisms, into their components.
• Gentamicin consists of 3 closely related components of the formula ##STR9##
• Gentamicin contains about 33% of C 1 , 41% of C 2 and 25% of C 1a .
• gentamicin is treated with acetic anhydride in water, whilst cooling, all the primary amino groups being acylated.
• the secondary amino groups are then converted into the corresponding urethane groupings with n-butyl chloroformate in aqueous ethanol.
• a mixture of compounds is thus obtained which, on a laboratory scale, can be separated by stepwise liquid/liquid extraction in a separating funnel.
• aqueous ammonium hydroxide Concentrated, aqueous ammonium hydroxide is used as the aqueous phase, and the lower phase of the two-phase system which is formed when 7 parts by volume of methylene chloride, 0.4 part by volume of propan-2-ol and 1 part by volume of concentrated ammonium hydroxide are mixed is used as the organic phase.
• the gentamicin C 1 derivative passes into the organic phase and the other two gentamicin derivatives remain in the aqueous phase. If appropriate, the organic phase can also be washed, in countercurrent, with aqueous ammonia.
• the preparation of the present invention may suitably be carried out without the formation of chemical derivatives on any unprotected amino groups.
• a particularly interesting process variant consists in converting a crude amino-trisaccharide of the formula (I), e.g. sisomicin, which has been obtained by fermentation into the corresponding 1,3,2',6'-tetra-N-acyl or -sulphenyl derivative, preferably acetyl derivative, of the formula (II), alkylating, preferably ethylating, the 3"-methylamino group, if appropriate isolating this derivative in a pure form, preferably by counter-current extraction, subsequently demethylating the resulting tertiary 3"-amino group, thereafter, if appropriate, providing the 3"-alkylamino group with a protective group and subjecting the product to the extraction according to the invention.
• This tertiary amine is not subjected to oxidative demethylation with an oxidising agent, for example potassium hexacyanoferrate-III in aqueous methanol, in the presence of a base, for example sodium hydroxide, the secondary 3"-ethylamino compound being formed.
• an oxidising agent for example potassium hexacyanoferrate-III in aqueous methanol
• a base for example sodium hydroxide
• the tertiary amine obtained as described above can be purified by continuous countercurrent extraction.
• the pure 1,2',3,6'-tetra-N-acetyl-3"-N-ethyl-sisomicin then obtained can thereafter be subjected to oxidative dealkylation as described.
• One of the preferred processes for the preparation of the crude 3"-N-alkylated derivatives of the 4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols of the formula (II) which contain amino-protective groups in all the positions except for position 3" consists in reacting the corresponding tetra-N-protected compounds, or acid addition salts thereof, with an aldehyde in the presence of a hydrogen donor reducing agent and working up the batch in a manner which is in itself known.
• This process in which the 3"-amino group in a 4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol reacts with an aldehyde and is simultaneously reduced in situ, is usually carried out at room temperature in the presence of air, although it can be more favourable to carry out the reaction under an inert gas (argon or nitrogen).
• the reaction usually goes to completion very rapidly, frequently in less than 60 minutes, which can be established by determinations by thin layer chromatography.
• Hydrogen donor reducing agents which are used in this process include dialkylaminoborane (for example dimethylaminoborane, diethylaminoborane and, preferably, morpholinoborane), tetraalkylammonium cyanoborohydrides (for example tetrabutylammonium cyanoborohydride), alkali metal borohydrides (for example sodium borohydride) and alkali metal cyanoborohydrides (for example lithium cyanoborohydride and sodium cyanoborohydride).
• dialkylaminoborane for example dimethylaminoborane, diethylaminoborane and, preferably, morpholinoborane
• tetraalkylammonium cyanoborohydrides for example tetrabutylammonium cyanoborohydride
• alkali metal borohydrides for example sodium borohydride
• alkali metal cyanoborohydrides for example lithium cyanoborohydride
• the process is usually carried out in an inert solvent.
• the solvent can be an organic or inorganic solvent, in which the selectively protected 4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol and the other reagents are soluble and which as far as possible reduces or prevents side reactions under the reaction conditions.
• anhydrous aprotic solvents can advantageously be used (for example tetrahydrofuran if the reducing agent is morpholinoborane), a protic solvent is nevertheless usually employed.
• Suitable protic solvents are, for example, a C 1 -C 6 alkanol or water or an aqueous C 1 -C 6 alkanol or other solvent systems which contain water, such as aqueous dimethylformamide, aqeous hexamethylphosphoramide, aqueous tetrahydrofuran or aqueous ethylene glycol dimethyl ether.
• the process is usually carried out in a pH range from 1 to 11, and preferably at pH 4 to 8.
• Another process according to the invention for the preparation of the 3"-N-alkylated amino-trisaccharide derivatives consists of alkylation of the tetra-N-protected amino-trisaccharides in question, with a non-blocked 3"-methylamino group, with alkyl halides.
• R 1 has the abovementioned meaning and Hal denotes a halogen atom, such as a chlorine, bromine or iodine atom,
• dimethylformamide as the solvent in these reactions.
• 1 to 10 molar equivalents of alkylating agent are used and the reaction is carried out at pH values from about 5 to about 12.
• an auxiliary base here, in order to trap the hydrogen halide liberated during the reaction.
• appropriate bases are alkali metal hydroxides, alkaline earth metal hydroxides, alkali metal carbonates and alkaline earth metal carbonates, alkaline earth metal oxides and carbonates and oxides of heavy metals, such as lead carbonate and silver carbonate, as well as mercury oxide or silver oxide.
• all compounds which are stable under the reaction conditions and which are capable of trapping the hydrogen halide formed can be used as auxiliary bases.
• One of the preferred processes for splitting off the 3"-N-methyl group from the crude or extractively purified trisaccharides with a tertiary 3"-amino group which have been obtained as described above is oxidated demethylation with customary oxidising agents.
• oxidising agents are heavy metal salts, peroxides, halogens, halogen oxyacids and salts thereof, nitrogen oxides and molecular oxygen.
• Preferred oxidising agents are permanganates, manganates, manganese dioxide, chromium trioxide, bichromates, chromates, alkyl-chromates, chromyl chloride, selenium dioxide, cobalt-III salts, cerium-IV salts, potassium hexacyano-ferrate-III, copper oxide, lead oxide, mercury oxide, mixtures of hydrogen peroxide and iron-III salts, iron-II salts, selenium oxide, osmium tetroxide, vanadates, tungstic acid and/or chromic acid, lead tetraacetate, chlorine, bromine, iodine, hypochlorates, chlorites, hypobromates, bromates, periodates, dinitrogen monoxide, nitrogen dioxide and air. If molecular oxygen is used, noble metal
• Particularly preferred oxidising agents are manganese dioxide, potassium hexacyanoferrate-III and potassium permanganate.
• the splitting reaction is preferably carried out in the presence of a diluent which is inert under the reaction conditions, preferably one in which the reactants dissolve.
• a diluent which is inert under the reaction conditions, preferably one in which the reactants dissolve.
• Suitable diluents of the type mentioned are water or mixtures of water and methanol, ethanol, i-propanol, tetrahydrofuran, dimethylformamide, dioxane, pyridine, ethylene glycol dimethyl ether and acetone.
• the reaction is generally carried out at a pH value of 3 to 12, depending on the nature of the oxidising agent used.
• the pH value can be established by adding an appropriate acid or base. Those acids or bases which do not decompose the starting compounds or the end product and cause no decrease in the activity of the oxidising agents are to be used. Rather, it is desirable for them to increase the activity of the oxidising agents.
• Inorganic acids which can be used are, for example, hydrochloric acid or sulphuric acid
• organic acids which can be used are, for example, acetic acid or formic acid.
• appropriate bases are ammonium hydroxide, alkali metal hydroxides, alkaline earth metal hydroxides, alkali metal alcoholates and alkali metal and alkaline earth metal salts of carboxylic acids.
• the pH value can be established either before the start of the reaction or during the reaction.
• the reaction is generally carried out at temperatures of -30° C. to 100° C., preferably of -20° to 0° C.
• the reaction time is half an hour to 50 hours.
• the reaction is carried out under normal pressure.
• the splitting off of the 3"-N-methyl-groups is also achieved by reaction with chloroformic acid esters in the presence of auxiliary bases, the methyl group being replaced by the corresponding alkyl- or aryl-oxycarbonyl group and penta-N-blocked compounds being formed.
• the protective groups are split off from the amino groups in the customary manner.
• the sulphenyl groups can be split off either with nucleophiles, such as hydrogen sulphide, thiophenol or 2-mercaptobenzthiazole, if appropriate also in the presence of mineral acids, or by heating with inorganic bases, such as alkali metal hydroxides or alkaline earth metal hydroxides, if necessary under increased pressure.
• nucleophiles such as hydrogen sulphide, thiophenol or 2-mercaptobenzthiazole
• Acyl protective groups such as acetyl, ethoxycarbonyl or t-butoxycarbonyl, can be split off with aqueous alkali metal hydroxide or alkaline earth metal hydroxide or with acids, such as trifluoroacetic acid or boron trifluoride etherate, in organic solvents or mixtures of organic solvents in water.
• the end products obtained after splitting off the protective groups are isolated as free bases or in the form of pharmaceutically usable acid addition salts.
• the process according to the invention represents a substantial improvement.
• the process according to the invention is broadly applicable and gives products in high yields with relatively little technological effort.
• the crude products used in the following Examples can be obtained by a method wherein the corresponding fermentation medium is acidified and then filtered; calcium ions are then removed, if necessary, with oxalic acid.
• the solution is neutralised with aqueous ammonium hydroxide and treated with a cation exchanger resin (NH 4 .sup. ⁇ form), onto which the aminoglycosides are bonded; the exchanger is wased, and eluted with aqueous ammonium hydroxide.
• the crude solutions of aminotrisaccharide which are thus obtained are evaporated until the ammonia has been removed.
• the following Examples, 3,4,6 and 8 to 16 illustrate the process of the present invention, whereas the remaining Examples illustrate the pre-purification of the crude product, the removal of protective groups or the formation of chemical derivatives.
• the crude product obtained in 3.1 was dissolved in 840 ml of ethanol and 160 ml of water, and 185 g of sodium carbonate were then added.
• a solution of 185 ml of n-butyl chloroformate in 185 ml of acetone was added dropwise at 50° C., 1500 ml of ethanol were then added, the mixture was allowed to cool and the inorganic salts are filtered off.
• the filtrate was evaporated to a syrup in vacuo and the title compound was obtained as a crude product in the form of a brown-coloured syrup.
• the syrup obtained in 3.2 was dissolved in 750 ml of concentrated aqueous ammonia. Lipophilic impurities were extracted from this solution with two 350 ml portions of methylene chloride. The sisomicin derivative was then isolated by extracting the ammonia solution twenty times with in each case 1500 ml of the lower phase of the system methylene chloride (7 parts by volume), propan-2-ol (0.8 part by volume) and concentrated aqueous ammonia (1.0 part by volume).
• the pure product was heated under reflux with 880 g of Ba(OH) 2 ⁇ 8H 2 O in 1500 ml of water. After about 8 hours, the barium salts were separated off by precipitation with CO 2 and filtration. The filtrate was deionised with a basic ion exchanger resin ("Lewatit” MP 500, OH.sup. ⁇ form). The sisomicin base was removed from the solution by adsorption onto a cation exchanger resin ("Lewatit" CNP-LF, NH 4 .sup. ⁇ form). The resin adduct thus obtained was washed with water. The sisomicin base was then eluted with 5% strength aqueous ammonia.
• sisomicin sulphate was obtained as a colourless solid.
• the syrup was dissolved in 50 ml of the lower phase of the two-phase system n-butanol/ammonia (concentrated)/n-hexane (ratio of 4.8:5.0:0.2--in each case parts by volume) and the solution was thoroughly stirred with 50 ml of the upper phase of the same system.
• the upper phase was evaporated in vacuo and the syrup thus obtained was heated under reflux with 40 g of barium hydroxide hydrate in 70 ml of water for 7 hours.
• Example 6.1 The residue from Example 6.1 was dissolved in 340 ml of concentrated aqueous ammonia. This solution was extracted 20 times with in each case 450 ml of a mixture of 340 ml of methylene chloride, which had been saturated with concentrated aqueous ammonia, and 135 ml of n-hexane, the tetra-N-methoxycarbonylsisomicin passing into the organic phase.
• the combined extract phases were freed from solvent in vacuo, the residue thus obtained was dissolved in 200 ml of concentrated aqueous ammonia and the solution was extracted 20 times with in each case 265 ml of a mixture of one part by volume of n-hexane and 2.5 parts by volume of methylene chloride, which had been saturated with concentrated aqueous ammonia.
• the combined extracts were freed from solvent in vacuo and the residue was dried until an amorphous solid was obtained.
• the resulting aqueous solution of the sisomicin base was introduced onto a column containing 370 ml of cation exchanger resin ("Lewatit" CNP, NH 4 + form), the sisomicin being bonded to the resin.
• the column was washed with 1000 ml of water and the sisomicin was then eluted with 5% strength aqueous ammonium hydroxide. After evaporating off the ammonia, sisomicin remained as a colourless solid. This was dissolved in 140 ml of water and the alkaline solution was adjusted to pH 4.8 with dilute sulphuric acid. The solution was stirred with active charcoal and filtered, and the filtrate was lyophilised.
• the solution obtained after separating off the exchanger resin was evaporated in vacuo.
• the residue was dissolved in 160 ml of ethanol and 30 ml of water, 45 g of sodium carbonate were added, and a solution of 45 ml of n-butyl chloroformate in 45 ml of acetone was added dropwise at 50° C. in the course of about 1.5 hours, whilst stirring.
• the mixture was allowed to cool, 200 ml of toluene were added and the solvents were distilled off in vacuo.
• the residue was taken up in 300 ml of ethanol and the mixture was stirred thoroughly.
• the undissolved material was filtered off and rinsed with ethanol and the combined filtrates were evaporated in vacuo.
• the syrup obtained after evaporating the extract phase consisted of a mixture of 1,2',3,6'-tetra-N-acetyl-3"-N-(n-butoxycarbonyl)-gentamicin C 1a , 1,2',3,6'-tetra-N-acetyl-3"-N-(n-butoxycarbonyl)-gentamicin C 2 and 1,2',3-tri-N-acetyl-3",6'-di-N-(n-butoxycarbonyl)-gentamicin C 1 .
• Gentamicin-C base was thus obtained as a colourless solid, which was converted into the sulphate salt by treating the aqueous solution with sulphuric acid and then lyophilising the mixture.
• the residue was dissolved in 100 ml of concentrated ammonium hydroxide. This solution was extracted with 150 ml of the lower phase of a system of 7 parts by volume of methylene chloride, 0.4 part by volume of propan-2-ol and 1 part by volume of concentrated ammonium hydroxide. The gentamicin C 1 derivative was thus transferred into the extraction agent, and the C 1a and C 2 derivatives remained in the ammonium hydroxide phase.
• the methylene chloride phase was extracted by shaking twice more with in each case 100 ml of concentrated ammonium hydroxide. It was then evaporated in vacuo and dried to constant weight.
• Gentamicin C 1 base was thus obtained as a colourless solid.
• the mixture was adjusted to pH 4.5 with 50% strength sodium hydroxide solution and was then allowed to come to room temperature and was diluted with 430 ml of ethanol. 82 ml of acetaldehyde were added to this solution at 0° and the mixture was stirred at room temperature for about 30 minutes.
• the ethyl compound was isolated, by extraction, from the concentrate obtained according to Example (11a). 1.4 liters of concentrated ammonium hydroxide were added to the concentrate and the mixture was initially extracted, in a 6 liter separating funnel by stirring with a blade stirrer (750 revolutions/minute), three times with in each case 180 ml of the lower phase of a mixture of methylene chloride/propane-1-ol/NH 4 OH, in a ratio of 7:0.5:1 (in each case parts by weight), the lipophilic impurities being separated off. The lower phases were in each case discarded; the aqueous phase contained the title compound.
• the aqueous phase was extracted by stirring 15 times with in each case 700 ml of the lower phase of the mixture of methylene chloride/propan-1-ol/NH 4 OH--in a ratio of 7:1.5:1 (parts by volume)--by following the procedure described above.
• the mixture was adjusted to pH 7 with 30% strength sulphuric acid, whilst stirring and cooling, and a solution of 30 g of copper sulphate in 55 ml of water was then added in order to precipitate copper hexacyanoferrate.
• the mixture was allowed to come to room temperature, whilst stirring, the precipitates were removed by centrifugation and were washed out 4 times with water, the combined centrifugates were adjusted to pH 7 with 50% strength sodium hydroxide solution and the mixture was evaporated into a thin syrup in vacuo (temperature: about 50° to 60° C.).
• the syrup was taken up in 40 ml of methanol, and 80 ml of acetone and 40 ml of ethyl acetate were successively added, whilst stirring thoroughly.
• the solution was adjusted to pH 11 with saturated methanolic sodium hydroxide solution. 0.2 ml of methyl chloroformate in 0.4 ml of acetone were then added dropwise in the course of 5 minutes, whilst stirring thoroughly, the mixture was subsequently stirred for a further 30 minutes and adjusted to pH 11 as above, and the addition of the chloroformate and the regulation of the pH were repeated a further three times in the manner described.
• 1,2',3,6'-Tetra-N-acetyl-sisomicin was converted completely into 1,2',3,6'-tetra-N-acetyl-3"-N-methoxycarbonyl-sisomicin, whereas, because of the comparatively lower reactivity of the 3"-ethylamino group, the 3"-N-demethyl-3"-N-ethyl compound had not reacted.
• the syrup thus obtained consisted of 1,2',3,6'-tetra-N-acetyl-3"-N-demethyl-3"-N-ethyl-3"-N-(n-octyloxycarbonyl)-sisomicin, as the main product, and contained 1,2',3,6'-tetra-N-acetyl-3"-N-methoxycarbonylsisomicin, as a by-product.
• the 3"-N-demethyl derivative which had been highly lipophilised in the manner described above, was then separated off in a pure form by extraction.
• the syrup was taken up in 80 ml of water and the 3"-N-(n-octyloxycarbonyl)-derivative was extracted quantitatively into the upper phase by stirring with 40 ml of n-butanol and 48 ml of n-hexane (about 10 minutes). The derivative was separated off and extracted by stirring twice with in each case 20 ml of water. The upper phase was evaporated to a syrup in vacuo. The combined lower phases, which contained the 3"-N-methoxycarbonylsisomicin, were worked up to give sisomicin.
• the syrup resulting from evaporating the upper phase obtained as described in Example (11d) was taken up in 40 ml of water and, after adding 24 g of barium hydroxide 8H 2 O, the mixture was heated for 5 to 6 hours (bath temperature: 150° C.). The octanol liberated was thereby distilled off (monitoring of the reaction by TLC in system E). The mixture was allowed to cool to about 100° C., the barium ions were precipitated by adding 30% strength sulphuric acid (dropwise) until the pH was 5 and the mixture was allowed to come to room temperature.
• the barium sulphate was removed by centrifugation, the centrifugate was extracted by shaking with 50 ml of methylene chloride or ethyl acetate and the aqueous phase was stirred with 1 g of active charcoal.
• the charcoal was filtered off and the filtrate was stirred with about 120 ml of the ion exchanger resin "Lewatit" MP 500, OH.sup. ⁇ form, until the pH was 11, by which means 3"-N-demethyl-3"-N-ethylsisomicin was converted into the free base.
• the ion exchanger was filtered off and rinsed with water.
• lipophilic impurities were separated off.
• the extraction agent phase which contained the lipophilic impurities, was evaporated in vacuo in an evaporator unit, and the solvent mixture obtained after condensation was adjusted to the original mixture values in a stock vessel. It was then recycled to the process as the extraction agent.
• the raffinate phase which had been freed from the lipophilic impurities, was passed continuously to a second ARD extractor unit with a nominal width of 150 mm, and was passed in countercurrent with 90 liters per hour of a solvent consisting of a mixture, which had been saturated with concentrated aqueous ammonium hydroxide, of 7 parts by volume of methylene chloride and 2 parts by volume of propan-2-ol.
• the aqueous-ammoniacal raffinate phase was taken off at the top end of the column. It had a residual content of 1,2',3,6'-tetra-N-acetyl-3"-N-ethyl-sisomicin of about 0.1% by weight and contained, above all, the hydrophilic impurities.
• the extract phase about 107 kg per hour, removed at the bottom of the column contained 0.615% by weight of 1,2',3,6'-tetra-N-acetyl-3"-N-ethylsisomicin, corresponding to 658 g of product per hour.
• the extract phase was concentrated in vacuo. The solvent evaporated off was recovered and employed again in the process. The overall yield in this extraction process was about 97% of 1,2',3,6'-tetra-N-acetyl-3"-N-ethylsisomicin.
• the product was 95% pure.
• the mixture was subsequently stirred for a further 45 minutes. It was allowed to come to 0° C., 5 g of sodium sulphite were added, the mixture was allowed to come to room temperature and 500 ml of methanol and 240 g of anhydrous sodium carbonate were successively added. The mixture was warmed to 40° C., whilst stirring, and 700 ml of isopropyl chloroformate in 300 ml of acetone were then added in the course of 3 hours. After stirring the solution at 40° C. for 15 hours, it was evaporated in vacuo until the methanol had been removed.
• the title compound was obtained by extraction from the mixture which contained 1,2',3,6'-tetra-N-acetyl-3"-isopropoxycarbonyl-sisomicin and was prepared as described in Example 14.
• 200 g of the syrupy product mixture prepared according to Example 14 were dissolved in 1.5 liters of 25% strength aqueous ammonium hydroxide.
• This solution was extracted 12 times with in each case 3 liters of the lower phase of a mixture of 7 parts by volume of methylene chloride, 0.6 part by volume of propan-2-ol and 1 part by volume of concentrated ammonia.
• the combined extracts were evaporated in vacuo and the residue was again subjected to the extraction process described above.
• the protective groups could be split off as described in Example 16. The sisomicin thereby obtained could then be re-used.
• the solution obtained according to 20.1 was extracted 25 times with in each case 322 g of the lower phase of a mixture (being in phase equilibrium) of 7 parts by volume of methylene chloride, 4 parts by volume of propan-2-ol and 4 parts by volume of concentrated aqueous ammonia.
• the combined extracts were freed from solvents in vacuo, 1,2',3,3",6'-penta-N-acetyl sisomicin being obtained as a colourless residue.
• the solution obtained according to 21.1 was extracted 25 times with in each case 322 g of the lower phase of a mixture (being in phase equilibrium) of 7 parts by volume of methylene chloride, 3 parts by volume of propan-2-ol and 3 parts by volume of concentrated aqueous ammonia.
• the combined extracts were freed from solvents in vacuo, 1,2',3,3",6'-penta-N-acetyl-gentamicin-C being obtained as a colourless residue.

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• 1980
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